The main goal of the present proposal is to investigate Na-K-Cl cotransport of vascular endothelial cells with respect to its role in endothelial cell function. The endothelium is known to play a central role in maintenance of vascular homeostasis. In this regard, endothelial cells have been shown to participate in regulating vascular processes such as thrombosis, vasoconstriction, and smooth muscle cell proliferation. The endothelium also provides a vitally important selective permeability barrier between the blood and interstitium. Modulation of the endothelial barrier permeability to macromolecules such as albumin has been shown to be a major determinant of fluid distribution between vascular and extravascular spaces. Thus, the functional importance of the endothelium has been well documented. However, little is known about the involvement of ion transporters in endothelial cell function. Previous studies from the applicant's laboratory have established that endothelial cells exhibit a prominent Na-K-Cl cotransport system that is regulated by a variety of vasoactive agents. These findings suggest that Na-K-Cl contransport may play an essential role in normal endothelial cell function. Two physiological roles have been demonstrated for Na-K-Cl cotransport in other cell types: volume regulation in both nonepithelial and epithelial cells; and vectorial transport of ions across epithelia. As an initial approach to determining the functional significance of the prominent Na-K-Cl cotransport system in endothelial cells, the proposed project will investigate the involvement of the cotransporter in endothelial cell volume regulation. Cell volume regulation has been hypothesized to be critical for maintenance of the endothelial permeability barrier. Thus, the influence of endothelial cell volume on macromolecular permeability of the barrier will also be evaluated. Specifically, the present proposal has two primary aims: 1) to test the hypothesis that Na-K-Cl cotransport participates in endothelial cell volume regulation; and 2) to test the hypothesis that alteration of endothelial cell volume influences endothelial permeability to macromolecules. In these studies, Na-K-Cl cotransport activity and intracellular volume of a variety of cultured endothelial cells will be evaluated. Macromolecular permeability will be assessed for albumin using two systems: 1) an in vitro system of endothelial monolayers grown on microporous membrane cell culture chamber inserts; and 2) an in vivo system of perfused hamster mesentery microvessels. The proposed studies should provide information regarding the role of plasma membrane ion transport systems in endothelial cell function. This information could lead to new therapeutic approaches to the treatment and prevention of vascular pathophysiological conditions such as hypertension and atherosclerosis.